Nonexponential kinetics of a single tRNAPhe molecule under physiological conditions - PubMed (original) (raw)

Nonexponential kinetics of a single tRNAPhe molecule under physiological conditions

Y Jia et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The fluorescence decay functions of individual, specifically labeled tRNAPhe molecules exhibit nonexponential character as a result of conformational dynamics occurring during the measurement on a single molecule. tRNAPhe conformational states that interchange much more slowly are evidenced by the distribution of lifetimes observed for many individual molecules. A structural model for the nonexponential decay indicates that the tRNAPhe-probe adduct fluctuates between two states, one of which provides conditions that quench the probe fluorescence.

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Figures

Figure 1

Fluorescence images of individual 1:1 tRNAPhe–TMR adducts. The tRNAPhe–TMR adducts are attached to the surface of amino-propyl-silanized glass in aqueous buffer (50 mM Tris, pH 7.6/50 mM KCl/10 mM MgCl2/2.5 mM dithiothreitol).

Figure 2

A sudden single-step bleaching of the 1:1 tRNAPhe–TMR adduct. This result is typical of the criterion used for each measurement to confirm that the signal originated from a single molecule. In this example, the molecule bleached after approximately 30 sec.

Figure 3

Fluorescence decay of single TMR molecules in the 1:1 tRNAPhe–TMR adduct (Upper) and in the polymethylmethacrylate film (Lower). The nonexponential decay kinetics of the tRNAPhe–TMR adduct is evident when the signal is compared with the single exponential decay of the fluorescence of rhodamine in a polymer film. PMMA, polymethylmethacrylate.

Figure 4

Probability distributions of fluorescence lifetimes (Upper) and amplitudes (Lower) observed from 50 single molecules. Each measurement satisfied the criteria of single-step bleaching.

Figure 5

Simulated structure of the 1:1 tRNAPhe–TMR adduct. TMR is yellow, s4U pink, and G15 green.

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Nonexponential kinetics of a single tRNAPhe molecule under physiological conditions - PubMed (original) (raw)